Method and medium for detecting the presence or absence of methicillin resistant staphylococcus aureus in a first generation biological test sample

ABSTRACT

A presence/absence test for  Staphylococcus aureus  ( S. aureus ) involves placing a first generation test sample in a solution that will clot in the presence of  S. aureus . The solution contains components that will selectively grow  S. aureus  and also contains clotting factors that will react with  S. aureus , if  S. aureus  is present in the sample, to clot the solution. Examples of specimen samples that can be tested include nasal swabs and lesion swabs, among others. The test can also be modified to detect the presence or absence of methicillin resistant  S. Aureus  (MRSA).

This application is a divisional of U.S. patent application Ser. No.12/356,847 filed Jan. 21, 2009, which claims the benefit of U.S.Provisional Patent Application Ser. Nos. 61/062,144, filed Jan. 24,2008; and 61/108,722, filed Oct. 27, 2008, all of which herebyincorporated by reference into the present application in theirentirety.

BACKGROUND OF THE INVENTION

1. Technical Information

The present method and test mixture relates to the detection ofStaphylococcus aureus in a biological, environmental, or food sample,and more particularly to those methods and test mixtures utilizingreacting factors which the target microbe(s) can convert into a clot.

2. Background Information

Staphylococcus aureus (S. aureus) can be a virulent pathogen of animalsand humans Moreover, it can cause severe food poisoning by theproduction of a toxin. Diseases caused by S. aureus cover a very wideclinical spectrum, from simple skin infections to life threateninginfections of the bones, heart, and organs. Of particular concern is therecognition that S. aureus infection is common after surgery. It is alsoassociated with intravenous tubing and other implants.

The bacterium S. aureus may be transmitted between healthy individualsby skin to skin contact, or from a commonly shared item or a surface(e.g., tanning beds, gym equipment, food handling equipment, etc.) wherethe transfer may be made to a subsequent person who uses the shared itemor touches the surface. Of great medical concern is the recognition thathealthy people entering hospitals may “carry” S. aureus (e.g., on theirskin, or in their noses, etc.) without any signs or symptoms that theydo so. In the presence of favorable conditions (often found in but notlimited to hospitals), the S. aureus can activate and cause seriousinfection. In addition, S. aureus can also be a source of foodpoisoning, often caused by a food handler contaminating the food product(e.g., meat, poultry, eggs, salads containing mayonnaise, bakeryproducts, dairy products, etc.)

There are two categories of S. aureus based on an individual clone'ssusceptibility to the class of antibiotics that began with methicillin.These are methicillin susceptible S. aureus (MSSA), and methicillinresistant S. aureus (MRSA). Until only a few years ago, MRSA was mostcommonly found in hospitals. Now, many are also present in the noses,skin, etc. of people in the non-hospital community. Moreover, these MRSAare increasingly causing serious infections in the community. MRSA isparticularly serious because very few antibiotics (e.g., vancomycin)have been shown to be uniformly effective against MRSA.

The Center for Disease Control and Prevention actively surveys for thedevelopment of methicillin resistant S. aureus. In 2000, the Society forHealthcare Epidemiology of America guidelines recommended contactisolation for patients with MRSA. In addition to the morbidity andmortality caused by MRSA, it has been estimated that each case ofinfection costs at least $23,000. Accordingly, many hospitals andnursing homes proactively sample patients for MRSA [Clany, M., ActiveScreening in High-Risk units is an effective and cost-avoidant method toreduce the rate of methicillin-resistant Staphylococcus aureus infectionin the hospital, Infection Control and Hospital Epidemiology27:1009-1017, 2006].

Meyer et al. (U.S. Pat. No. 4,035,238) describes the use of a broth forthe detection of S. aureus that utilized mannitol as a source of carbonand DNA methyl green as an indicator. Neither of these chemicals arecoagulase reactive substrates.

Rambach (U.S. Pat. No. 6,548,268) employs at least two chromogenicagents in an agar medium: 5-bromo-6-chloro-indoxyl-phosphate; and5-bromo-4-chloro-3-indoxyl glucose in the presence of deferoxamine. Anindividual colony hydrolyzing these substrates will produce colors thatwill mix with each other and not be independent of one another.

A large number of classical culturing procedures are utilized to detectMSSA and MRSA from human, animal, food, etc. samples. They have incommon a basic medium with chemical inhibitors such as 6-8% sodiumchloride, potassium tellurite, and a variety of antibiotics. For exampleStevens and Jones described the use of a trehalose-mannitol-phosphataseagar [Stevens, D L and Jones, C. Use of trehalose-mannitol-phosphataseagar to differentiate Staphylococcus epidermidis and Staphylococcussaprophyticus from other coagulase-negative staphylococci, J. of Clin.Microbiology 20:977-980, 1984]. The use of mannitol as a carbon sourceand salt as a selective agent into an agar known as mannitol-salt agarhas been commonly used in clinical laboratories [Baird, R. M. and W. H.Lee, Media used in the detection and enumeration of Staphylococcusaureus, Int. J. Food Microbiology. 26:209-211, 1995]. Within the priorart of culturing, it is generally accepted procedure to performcoagulase tests utilizing samples of S. aureus that are isolated in apure culture.

The procedure “S. aureus ID” [Bio Merieux, La Balme Les Grottes, France]uses an alpha-glucosidase substrate in agar to detect S. aureus. Asingle substrate is utilized. [Perry, J. D. et al., Evaluation of S.aureus ID, a new chromogenic agar medium for detection of Staphylococcusaureus, J. Clin. Microbiology 41:5695-5698, 2003]. A variant of thismedium, which contains added antibiotics and sodium chloride, isdesigned to detect MRSA [Perry et al., Development and evaluation of achromogenic agar medium for methicillin-resistant Staphylococcus aureus,J. of Clin. Micro. 42:4519-4523, 2004].

Selepak and Witebsky disclose a study evaluating the inoculum size andlot-to-lot variability of the tube coagulase test for S. aureus.Specimens were collected and isolates were generated from the bacterialcolonies on agar plates. Tubes containing anticoagulated rabbitcoagulase plasma were inoculated with a part of, or more than one,staphylococcal colony from the isolates. The tubes were incubated andexamined for the presence of clot. According to Selepak and Witebsky,“with some isolates and some lots of coagulase plasma, even a singlecolony [from the isolate] may not provide enough inoculum for a positivecoagulase test”. Furthermore, Selepak and Witebsky state that“[e]xpressed more quantitatively, at least 10⁸ organisms per ml shouldbe used whenever possible for each coagulase tube test. Our data furthersuggest that S. aureus does not grow in coagulase plasma; therefore, theincubation of coagulase plasma for 18 to 24 h does not compensate forthe use of small inoculum.” Thus, Selepak and Witebsky indicate that itis impractical, if not impossible, to detect the presence or absence ofS. aureus in first generation biological specimen samples using a directcoagulase test.

[Selepak, S. T et al, “Inoculum Size and Lot-to-Lot Variation asSignificant Variables in the Tube Coagulase Test for Staphylococcusaureus”, Journal of Clin. Microbiology, November 1985, p. 835-837]

It would, therefore, be desirable to provide a test mixture and a methodthat can rapidly detect S. aureus directly from a sample, one that doesnot require a skilled technician to perform the method, one that can beperformed without the need to develop isolates from the specimen (i.e.,one that can be performed on a “first generational” sample), and onethat does not require a large concentration of S. aureus organisms to beaccurate.

SUMMARY OF THE INVENTION

This invention relates to a method and test mixture for specificdetection of S. aureus bacteria in a biological, environmental, or foodsample. In the detection of S. aureus, a test mixture (which mixture mayalso be referred to as a “medium”) is utilized that includes coagulasesubstrates (sometimes referred to as “coagulase reacting factors”) thatreact specifically with coagulase produced by S. aureus to form a clot,admixed with constituents that facilitate the multiplication of S.aureus (also referred to as “growth promoting constituents”). Hence, thepresent method and test mixture utilize coagulase substrates that areactivated by the coagulase produced by S. aureus, and the enzymecoagulase is specific to pathogenic staphylococci, as is disclosed inthe Code of The Federal Register, Title 21, Chapter 1, Sub Part C, Sec.866.2160 “Coagulase Plasma”. Inhibitors and antibiotics may be includedto inhibit or otherwise negatively affect competing bacterial growth,but are not required. The untreated sample (e.g., collected from a nasalswab from a person, or off of a surface, etc.) is added to the testmixture, and the inoculated test sample is incubated. If S. aureus ispresent within the sample, the S. aureus will multiply within the testmixture and will produce coagulase that reacts with the coagulasesubstrates. The reaction between the coagulase produced by the S. aureusand the coagulase substrates within the test mixture will produce adetectable clot within the test mixture in a time period typicallybetween two and twenty-four hours, positively indicating the presence ofS. aureus.

Under the present method, the clot may be dissolved to release viable S.aureus into a liquid, which liquid can then be subjected to furtheranalyses, including but not limited to: antibiotic susceptibility tests,molecular fingerprinting, genetic analysis, etc. As will be describedbelow in greater detail, the antibiotic cefoxitin or an inducer of themecA gene may be included within the test mixture to enable methicillinresistant S. aureus (MRSA) specific testing within the firstgenerational sample, or the dissolved clot mixture may be tested toascertain the presence or absence of MRSA therein.

The test mixture is preferably prepared in a form that facilitateshandling, packaging, storing, etc., of the test mixture. A dry powderthat can be hydrated into liquid form is a particularly preferable formfor the test mixture, but the present invention is not limited to apowder form. The test mixture may assume a liquid form, or any otherform (e.g., paste, gel, etc.), preferably one that can be hydrated foruse.

The coagulase substrates within the test mixture may be provided withinplasma, or may be provided by another substance that is operative toreact with the coagulase produced by S. aureus to form a clot. Presenttesting indicates that rabbit plasma is a favorable source of acoagulase substrate. Other plasmas (e.g., pork plasma) may be usedalternatively. Fibrinogen is another example of a source of a coagulasesubstrate. In those embodiments that utilize plasma as a source of acoagulase substrate, it may be preferable to add a non-plasma source ofa coagulase substrate to the test mixture to ensure an adequate sourceof coagulase substrate within the test mixture. As an example, ourtesting indicates that combining fibrinogen and rabbit plasma within thetest mixture is an effective means for ensuring a consistent, adequatesource of coagulase substrates. An advantage of adding a material suchas fibrinogen to the test mixture is that it increases the performanceconsistency of the test mixture, and makes the method less susceptibleto variability that may occur with plasma.

The growth promoting constituents within the test mixture thatfacilitate the multiplication of and sustain S. aureus can be varied tosuit the application. Those in the art will recognize that manydifferent combinations of constituents, and varying relative amounts ofthe same constituents, can be used to provide the same functionality.Growth promoting constituents include sources of nitrates and proteins,material operative to assist in the generation of nucleic acidsynthesis, sources of energy for the S. aureus, sources of amino acidgrowth factor, and in some embodiments materials operable to help repairdamaged target organisms. This list of growth promoting constituentsdoes not represent all of the materials that can be beneficial withinthe test mixture, but does illustrate materials that are acceptable(e.g., vitamins, salts, minerals, inorganic moieties, etc.). The testmixture may include other constituents that benefit the performance ofthe test mixture.

In most applications of the present invention, it will be desirable toutilize a test mixture that includes the following: a) an effectiveamount of amino acids; b) an effective amount of nitrogen sources; c) aneffective amount of salts; d) an effective amount of vitamins; and e) aneffective amount of calcium. Those skilled in the art will recognizethat natural sources of such amino acids can be used rather than puresources. The natural sources (e.g. extract of whole organisms, such asyeast) may be in mixture fowl or in purified form. The natural mixturescan contain varying amounts of such amino acids and vitamins. Thoseskilled in the art will further recognize that many differentcombinations of amino acids and vitamins can be used in presentinvention test mixture.

Those in the art will further recognize that carbon, nitrogen, traceelements, vitamins, amino acids and selective agents can be provided inmany forms. Generally, it is preferred to have an amount of vitamins andamino acids within a predetermined range, but those in the art willrecognize that the actual properties of each ingredient may be varied sothat reduction in the amount of one ingredient can be compensated by anincrease in the amount of another. This is particularly relevant whenthe essential amino acids, trace elements or vitamins of the microbessought to be detected are known. Some ingredients may be provided inreduced amounts or deleted if they may be synthesized endogenously bythe microorganism whose presence is to be determined. Salts may beprovided as a source of ions upon dissociation.

The test mixture may be packaged in a container (e.g., a test tube, acontainer with a flat bottom wall, etc.) that facilitates the testingprocess. If the medium is prepared in a form that can be hydrated, themixture can be hydrated with sterile water or non-sterile water.

To detect the presence of MSSA or MRSA within a sample, the sample isobtained from a biological, environmental, or food specimen. A samplecollected using a nasal swab is an example of a first generation samplethat is particularly convenient to collect and test using the presentinvention. Once collected, the sample is inoculated into the testmixture.

The inoculated sample is incubated under conditions favorable tofacilitate the multiplication of any S. aureus that may be presentwithin the inoculated sample. In the case of a powdered test mixturehydrated with water, the incubation may be carried out at temperaturesbetween about 20° C. to 42° C. The combination of sequential enzymespecificity, S. aureus enhancing growth factors, and antibioticselectivity provides multiple hurdles which prevent the competingnon-target bacteria from being detected within the test period; e.g. 24hours or less.

The present invention test and method can be used in hospitaladmissions, routinely in intensive care units, in nursing homes,dialysis patients, people receiving home immunosuppressive therapy, andthe like. It can also be used in environmental settings (e.g., gyms,tanning salons, restaurants, etc.) where the bacteria S. aureus may betransferred from a human carrier and it can be used to test variousdifferent foods for S. aureus contamination. It will be appreciated thata substantial benefit of the present method and mixture is that they maybe performed/used without the need for expensive equipment or skilledmedical technologists. Another substantial benefit of the presentmethod/mixture is that it is operable with a relatively small amount ofS. aureus within the test sample; e.g., the present method/mixture hasdetected S. aureus in samples having concentrations of S. aureus as lowas 100 CFU/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

Details of the invention will become more readily apparent from thefollowing detailed description of several embodiments of the inventionwhen taken in conjunction with the accompanying drawings in which:

FIG. 1 is a side elevational view of a test tube containing a powderculturing mixture which is formulated to detect the presence or absenceof S. aureus in a first generation biological sample of a nasal swab;

FIG. 2 is a side elevational view of the test tube of FIG. 1, butshowing the culturing mixture having been hydrated by water;

FIG. 3 is a side elevational view of the test tube FIG. 2 and showing acotton swab inserted into the test tube to deposit a first generationbiological specimen nasal swab in the medium;

FIG. 4 is a side elevational view of the test tube of FIG. 3 after thespecimen has been deposited and cultured in the medium for a period oftime and indicating the absence of S. aureus in the specimen; and

FIG. 5 is a side elevational view similar to FIG. 4 but showing the testtube medium after the culturing period and indicating the presence of S.aureus in the specimen.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

FIG. 1 is a side elevational view of a test tube denoted by the numeral2 which preferably has a flat bottom 4 and a top closure 3, and whichcontains a dry powdered test mixture 1 which is formed in accordancewith this invention for detecting the presence or absence of S. aureusin a sample; e.g., a first generational biological sample. The tube 2 isalso provided with a reference line 5 which indicates the amount ofwater to be added to the tube 2 in order to properly hydrate thepowdered mixture 1 for specimen sample testing.

Acceptable hydrated test mixtures can be made using the followingconstituents in the ranges indicated, to create 15 ml of test mixture:

Specific Examples of Range of Constituent Constituent Quantities perQuantities per 15 ml Test Constituent 15 ml of Test Mixture MixtureNitrate Broth 7.5 ml 1.0 ml-9.0 ml  Water 7.5 ml 1.0 ml-9.0 ml  Uracil10.0 mg 1.0 mg-20.0 mg Sodium Pyruvate 10.0 mg 1.0 mg-20.0 mgL-glutamine 20.0 mg 5.0 mg-40.0 mg Sodium Sulfite 1.0 mg 0.5 mg-2.0 mg Rabbit Plasma 100.0 mg 15.0 mg-500.0 mg Fibrinogen 100.0 mg 15.0mg-500.0 mgThe specific example of the constituent quantities per 15 ml of testmixture provided above represents a particular test mixture formulationthat was tested and found to perform satisfactorily. This specificexample does not represent all test mixture formulations, and thepresent invention is not limited thereto. As stated above, those in theart will recognize that many different combinations of constituents, andvarying relative amounts of the same, can be used to provide the samefunctionality Hence, the present method and mixture contemplates that anumber of different constituent formulations can be made within theaforesaid ranges.

As noted in FIG. 2, the powdered mixture 1 is properly hydrated by theaddition of water, preferably distilled water, to form a hydrated testmixture 6 into which the sample (e.g., carried on a nasal swab) isdeposited.

First generational test samples can be collected by a variety ofdifferent techniques; e.g., a human sample can be collected by wiping aswab within the nose of a subject. Nasal swabs are a particularlyconvenient way of collecting a test sample, but they are not the onlycollection method; e.g., test samples can be collected from throatswabs, skin lesions, undamaged skin, etc. First generationalenvironmental samples can be collected by various known methods; e.g.,wiping or swabbing a surface using a dry or wet wipe/swab. Likewise,first generational food samples can be collected from the food itself,or wiping food residue from surfaces in contact with the food, etc. Oncethe sample is collected, it can be deposited in the hydrated testmixture 6; e.g., using the same cotton swab 8 which has been used togather the first generation sample from the source thereof Once thespecimen sample is deposited in the test mixture 6, it is incubatedwithin the test mixture for a period of time typically less thantwenty-four hours. The incubation may occur at any temperature that isacceptable under the circumstances. After the inoculation period, thecontainer (e.g., test tube 2) holding the inoculated test mixture can beinspected for the presence of a clot; e.g., the test tube 2 can betilted to one side as shown in FIGS. 4 and 5 to see if the meniscus 10of the text mixture will move or whether a clot keeps the test mixturebelow a reference line 5. The presence of a clot indicates that S.aureus is present in the test sample, and the absence of a clot in theinoculated test mixture indicates that S. aureus is not present in thetest mixture 6, as shown in FIG. 4. In some instances, the entireinoculated test mixture will clot, and in others some liquid will remainin the container with the clot. Approximately 80% of the present testsperformed using first generation nasal samples clotted within six hourswhen S. aureus is present in the first generation test sample.

To determine the effectiveness of the present method and mixture, acontrol study was performed involving sixty (60) control samples titredto contain varying amounts of MSSA, and sixty (60) control samplescontaining varying amounts of MRSA Standard clones of MSSA and MRSA weregrown in trypticase soy broth (TSB), and were diluted by log 10increments. The present invention test mixture was inoculated with a setamount (0.1 ml) of each the control samples. A first set of theinoculated test mixtures were incubated at 35° C., and second set of theinoculated test mixtures were incubated at 23° C. Of the sixty controltest samples, all were positive for S. aureus in five hours, forty-nine(49) were positive in four hours; thirty-six (36) were positive in threehours, and twenty-four (24) in two hours. Data detailing therelationship between the concentration of the inoculum, and incubationtemperature was as follows:

S. aureus CFU/ml Clot at 35° C. Clot at 23° C. 7 log 10 2.0 h 3.0 h 6log 10 3.0 h 3.0 h 5 log 10 4.0 h 4.0 h 4 log 10 6.0 h 7.0 h 3 log 1010.0 h  11.5 h  2 log 10 15.0 h  21.0 h The concentration of S. aureus within the clots were all at least 5 log10.

In addition to the above described control study, a clinical study wasperformed using fifty samples. The samples were taken from a medicalintensive care unit by culturing patient nares. The patients were notidentified, nor were the results of any “standard” culture available(FDA protocol). The samples were plated on mannitol salt agar (MSA)using swabs. After plating the samples on MSA, the swabs were used toinoculate the test mixture. Clotting was observed for each hour fortwenty-four hours. There were no false positives.

Number of Number of Number of samples samples detected samples detecteddetected positive for positive for positive for identified bacteria byidentified bacteria identified both MSA method and by present bacteriaby MSA Bacteria present method method alone method alone MSSA 9 2 — MRSA13 2 1When a clot was observed, a portion of the clot was removed anddissolved. A quantitative count of CFU/ml was performed from thedissolved clot material.

In some embodiments, the present method/mixture may include means todistinguish between MSSA and MRSA. For example, cefoxitin in aconcentration of about 10-100 mg/ml or another MecA gene inhibitor canbe included in the test mixture. Any MSSA present within the test samplewill be killed, but MRSA will not. Thus, if a clot does form, the S.aureus in the test sample will have been shown to be MRSA. If a clotforms and confirms the presence of MRSA, the clot can then be dissolvedin order to perform further analyses of the S. aureus bacteriumdetected.

It will be appreciated that the test of this invention is significantlysimpler to perform than the standard tests which are currently in use,as typified by the coagulase plasma procedure suggested by RemelProducts, Thermo Fisher Scientific, Lenexa, Kans., U.S.A. The Remelprocedure, which is approved by the FDA and appears in the Code of theFederal Register as an exempt test, requires a two step test for S.aureus wherein microbe colonies from the specimen are first grown in anagar medium and screened for suspected S. aureus colonies using a gramstain and catalase slide test before proceeding to a second coagulasetest step. There are complications relating to this type of coagulasetest, namely: 1) colonies for coagulase testing must not be picked frommedia containing high concentrations of salt as false positive resultsmay occur; 2) in the first step slide test procedure, theorganism/saline suspension must be observed for auto-agglutination priorto the addition of the coagulase plasma to prevent a false positive testreading; and 3) false negative coagulase reactions may occur if the testculture is older that 18-24 hours, of if there is scant growth.

While the invention has been described with respect to preferredembodiments, those skilled in the art will readily appreciate thatvarious changes and/or modifications can be made to the inventionwithout departing from the spirit or scope of the invention as definedby the appended claims.

1. A method for detecting the presence or absence of MethicillinResistant Staphylococcus Aureus (“MRSA”) in a first generationbiological test sample, the method comprising the steps of: a) providinga test mixture containing a source of powdered coagulase substrates, andan amount of cefoxitin effective to kill any Methicillin SusceptibleStaphylococcus Aureus (“MSSA”) in the test sample; b) hydrating the testmixture in a container; c) admixing said first generation biologicaltest sample with the hydrated test mixture in the container; d)incubating the admixture of the first generation biological test sampleand the test mixture in the container at temperatures in the range ofabout 20° C. to about 42° C.; and e) detecting the presence or absenceof MRSA in the admixture of the test sample and the hydrated testmixture based on the presence or absence of clotting in the admixture.2. The method of claim 1, wherein the test mixture includes rabbitplasma as a source of coagulase substrates.
 3. The method of claim 2,wherein the test mixture includes fibrinogen as a source of coagulasesubstrates.
 4. The method of claim 1, wherein the test mixture furtherincludes: a) an effective amount of amino acids to facilitate theproduction of protein; b) an effective amount of nitrogen sources tofacilitate the production of protein and nucleic acid; c) an effectiveamount of salts to facilitate the transport of nutrient into a cell; d)an effective amount of vitamins to facilitate the biochemical reaction;and e) an effective amount of calcium to facilitate the transport ofnutrient into a cell.
 5. The method of claim 1, further comprising thestep of gathering the first generation biological test sample byswabbing a nasal passage of a human subject.
 6. The method of claim 1,further comprising the step of gathering the first generation biologicaltest sample by swabbing an environmental surface.